This invention is in the general fields of biochemical engineering and veterinary medicine. More specifically, this invention relates to improved, controlled-release formulations containing human interleukin-2 (hIL-2) and derivatives thereof for administration to livestock, and a method for preventing shipping fever and other stress-related diseases in livestock by administration of compositions of the invention.
Livestock food animals, particularly cattle, are adversely affected by shipment and feedlot conditions, which involve stress from overcrowding, weaning, transport, sometimes severe weather, etc.: in general, an unnatural environment. One syndrome, commonly known as "shipping fever" or bovine respiratory disease syndrome (BRDS) is a complex of diseases rather than a specific disease. BRDS is characterized by immune suppression and propensity to succumb to infection by one or more viral or bacterial pathogens.
Other animals also exhibit adverse reactions to stress. For example, pigs can suffer disease and/or negative respiratory reactions to weaning, or even poor weather. Again, the etiology does not lend itself to experimental modeling. No general treatment for stress-related disorders in livestock has been found. Sick animals are typically treated with antibiotics. Recently, interferon preparations have been offered for treating shipping fever.
There is considerable background information available with respect to the biological activity of hIL-2. IL-2 may be prepared by recombinant methods or obtained from appropriate cell cultures, e.g., from the supernatant of concanavalin-A (ConA) stimulated spleen cells. There are several activities measurable in vitro. First, IL-2 is a T-cell growth factor, as measured by thymidine uptake in cultures of cytotoxic or helper T-cell lines (T.sub.c or T.sub.h) when IL-2 is added. It is mitogenic with respect to adult thymocytes, and stimulates a cytotoxic cell response in LAK (lymphokine-activated killer) cells. It has also been shown to replace T.sub.h cells in athymic murine spleen cell cultures (J. Watson et al, Immunological Rev (1980) 51:257-258). Specifically, in the presence of IL-2 and antigen, certain T.sub.h cells are stimulated which then are able to contribute to antibody responses. Presumably this occurs because IL-2 is involved in the antigen-dependent maturation of T.sub.h cells in these nude mouse spleen cultures.
IL-2 has also been shown to directly affect B cells in vitro. Both B cell proliferation and antibody secretion (IgM and IgG) are enhanced by IL-2 in populations of purified, activated B cells (M.C. Mingari et al, Nature (1984) 312:641; R. Mittler et al, J Immunol (1985) 134:2393-2399; A. Muraguchi et al, J Exp Med (1985) 161:181-197).
It is not clear how these in vitro activities translate into a specific in vivo mechanism for mounting an immune defense. However, with respect to in vitro studies, cross-reactivity among species of various IL-2s has been studied. For example, hIL-2 supports activated rabbit and mouse T lymphocytes to approximately the same extent as the endogenous rabbit or mouse IL-2 (D. Redelman et al, J Immunol Method (1983) 56:359-370). Human IL-2 behaves as a qrowth factor not only for human T-cells, but also peripheral blood lymphocytes or splenocytes from other primates, horse, guinea pig, cat, rat, and mouse (F.W. Ruscetti et al, Blood (1981) 57:379-393). Human IL-2 is also known to enhance the development and maintenance of bovine cytotoxic lymphocytes in vitro (J. Carter et al, Fed Proc (1985) 44:1290). Native hIL-2 and recombinant IL-2 exhibit the same range of activity on animal cells in in vitro lymphocyte proliferation studies (M.V. Doyle et al, J Bio Resp Mod (1985) 4:96-109).
Some in vivo data are also available. The administration of IL-2 in vivo has been shown to restore immunocompetence in nude mice in response to heterologous erythrocytes (H. Stotter et al, Eur J Immunol (1980) 10:719-722). There is some information concerning in vivo cross-species reactivity as well. Human IL-2 is able to reconstitute spleen cell responses in mice infected with a parasitic protozoan (S.G. Reed et al, J Immunol (1984) 133:3333), while in vivo injection of IL-2 of human origin stimulates the splenic T-cells in nude mice (J.J. Farrar et al, Immunol Rev (1982) 63:158).
In summary, it is known that IL-2 behaves in some manner in vivo to mediate a successful immune response, including a response to a specific antigen, and in vitro studies have shown that cross-species reactivity of hIL-2 is very diverse (prior in vivo cross-species studies have involved only murine subjects for hIL-2). However, because the mechanism of involvement of IL-2 in the immune response is not understood, it is not possible to predict the behavior of IL-2 in boosting an immune response to prevent or ameliorate a particular disease or to predict its overall effect. Accordingly, there is no suggestion in the art that IL-2, and in particular hIL-2, would successfully mitigate the incidence of shipping fever or other stress-related syndromes that affect livestock.
Additionally, administration of protein and polypeptide agents to livestock has proved to be quite problematic. As peptides and proteins are typically digested upon oral administration, such agents must be administered by parenteral means. Many biologically active peptides and proteins have extremely short halflives in serum, which necessitates frequent administration (e.g., b.i.d.) to maintain therapeutic levels of the drug. Although this is an acceptable administration regime for human subjects (although not necessarily preferred), it may be considered unacceptable to the rancher who must treat hundreds or thousands of animals. The business of growing livestock is highly competitive, and lends a keen sense of economy to the operator. Thus, any treatment program must provide a tangible benefit, for example, increased weight gain in animals, increased lean to fat ratio, increased survival rate, etc. Further, the benefits must outweigh the cost of treatment, including the cost of personnel to administer the treatment. As excessive handling is detrimental to livestock (intramuscular injection while an animal is restrained in a chute is quite stressful to the subject), these goals are best met by selecting an active agent which is inexpensive and administered only infrequently (preferably only once). Infrequent administration is also preferred for the reason that it minimizes the chance of infection, as such administration is typically not performed in an ideal, hygienic environment.
Accordingly, a peptide or protein agent should either have a long half-life in the serum, or should be administered using some form of controlled release device. Such devices as used for other veterinary agents are typically either membrane-type devices (having a central reservoir containing the active compound, surrounded by a rate-controlling membrane), or monolithic-type devices (typically a solid matrix, e.g., of silicone rubber, having the active compound dispersed throughout). Design of such devices must balance the factors of release rate, completeness of delivery, and induction period, as well as biocompatibility and acceptability for use in food animals.
The ideal controlled-release device would administer the active compound at a constant ("zero-order"), therapeutic rate, beginning at the moment of administration and continuing until 100% of the compound contained in the device had been released. Further, the device would not cause inflammation or other adverse effects, and would leave no residue. Frequently, such devices as are presently used (e.g., Compudose.RTM., a silicone rubber matrix impregnated with growth-promoting steroids) are administered to portions of the animal not used for food, e.g., in the ear cartilage, or in portions of the animal removed at slaughter as offal.
However, the characteristics of the ideal device are seldom attained. The release pattern of most devices consists of a high initial release rate, followed by a logarithmically declining release falling eventually to subtherapeutic levels. Typically as much as 20-40% of the active compound is retained in the implant and is never released. Caution must be exercised that the initial release does not provide toxic serum levels of the active compound. The amount of compound released in excess of the therapeutic level, and that which is retained in the device, is essentially wasted. This is a particularly egregious drawback when the active compound is an expensive peptide or protein agent. Further, even when the device is not especially inflammatory, a foreign-body reaction often ensues which results in the device's encapsulation in fibrous tissue. Such encapsulation impedes the drug administration, and degrades the quality of meat at the injection site.
One form of sustained-release delivery system is the microcapsule or microsphere. Microcapsules/spheres are essentially small particles of active compound embedded in a suitable polymer to form spheres ranging in diameter from about 40-500 um. Microcapsules of less than about 300 um (preferably &lt;150 um) are easily administered by injection when suspended in a suitable liquid vehicle. A large variety of polymers may be selected for use in microcapsules, although the particular polymer which is best suited for a particular application is often difficult to determine. The necessary considerations include interaction between the polymer and the active compound, the solubility of the compound in the polymer, the stability of the polymer and its rate of degradation (if any), its biocompatibility, the morphology of the resulting microcapsule as it degrades, etc. Microcapsule formulations encapsulating steroids and other agents are reported in the literature, for example, T.R. Tice et al, Pharm Tech (1984) 8:26-35; D.R. Cowsar et al, Meth Enzymol (1985) 112:101-116; and L.R. Beck et al, "Long Acting Steroid Contraception" (1983, Raven Press, NY, Ed. D. Mishell) pp. 175-199.